Replication-type oncolytic adenovirus that can suppress tumor progression and extend the survival time of tumor-bearing individuals and its applications

Abstract

The present invention relates to the field of tumor biotherapy, and specifically discloses a recombinant adenovirus or adenoviral vector expressing a glucagon-like peptide-1 (GLP-1) receptor agonist, which has shown unexpected anti-cancer effects and survival times in tumor-bearing subjects, and further discloses a pharmaceutical composition comprising the recombinant adenovirus or adenoviral vector.

Description

[Technical Field]

[0001] The present invention relates to the field of tumor therapy with recombinant adenoviruses or adenoviral vectors. [Background technology]

[0002] Cancer has become the number one disease threatening human life and health. In China, the number of cancer patients is increasing by approximately 4 million per year, and the number of cancer-related deaths is nearly 3 million. Surgery is the most effective treatment for early-stage cancer, but it does not have a significant therapeutic effect on many patients with advanced or metastatic cancer. Although adjuvant therapies such as radiation therapy and chemotherapy can inhibit tumor progression to some extent, the overall survival rate of cancer patients has not yet been significantly improved. New, safe, and effective therapeutic agents are now the focus of pharmaceutical research worldwide.

[0003] Tumor immunotherapy has attracted attention for its significant therapeutic effects. This treatment utilizes the body's immune system to kill cancer cells by activating and / or modulating immune pathways. Oncolytic viruses are a new trend in the field of tumor immunotherapy. The FDA's approval of the oncolytic virus T-Vec at the end of 2015 has led to increased emphasis on oncolytic virus-mediated anti-cancer immunotherapy. Oncolytic viruses are a type of virus that can self-replicate and proliferate in large numbers within cancer cells, and they possess numerous unique anti-cancer effects, including inducing immunogenic death of cancer cells, utilizing the body's immune monitoring, and expressing recombinant proteins to modulate the tumor microenvironment. Currently, clinical trials involving oncolytic viruses such as adenovirus, herpes simplex virus, measles virus, poxvirus, and vesicular stomatitis virus are underway, and favorable therapeutic effects have been observed.

[0004] However, solid tumors can escape immune monitoring through multiple mechanisms, including abnormal metabolism and an inflammatory microenvironment, facilitating their invasion and metastasis. Research has shown that oncolytic viruses activate the immune system, but also increase tumor-promoting inflammation in the tumor microenvironment and alter the metabolic microenvironment, adversely affecting their anti-cancer effects. The present invention discloses a novel oncolytic virus construct and its application in anti-cancer therapy. Summary of the Invention

[0005] One aspect of the present invention relates to a recombinant adenovirus or adenoviral vector comprising a first promoter, an E1A early activation replication element, an optional second promoter, and a target nucleotide (e.g., DNA) sequence encoding a target protein comprising a glucagon-like peptide-1 (GLP-1) receptor agonist, wherein the first promoter is a constitutive promoter and the optional second promoter is a constitutive promoter that is the same as or different from the first promoter, and when the target protein is GLP-1(7-36), the Ad5 adenovirus or adenoviral vector must comprise two promoters.

[0006] Another aspect relates to a pharmaceutical composition comprising said recombinant adenovirus or adenoviral vector and pharmaceutically acceptable auxiliary ingredients.

[0007] Another aspect relates to the application of the recombinant adenovirus or adenoviral vector and pharmaceutical composition thereof in anti-tumor treatment and / or prevention in a subject, which involves inducing an anti-tumor effect mediated by CD8+ T cells.

[0008] Another aspect relates to the production of said recombinant adenovirus or adenoviral vector. [Brief explanation of the drawings]

[0009] [Figure 1A]1 shows an example of a single-promoter shuttle vector used to produce a recombinant adenovirus or adenovirus vector expressing GLP-1 according to the present invention. [Figure 1B] An example of a DNA construct of the single-promoter shuttle vector described in Example I is shown, which is used to produce a recombinant adenovirus or adenovirus vector expressing a GLP-1 analog (e.g., AdV-GLP-1(7-37 / A8G)-CMV), where IL-2 SS is a DNA sequence encoding the IL-2 signal peptide, the amino acid sequence of which is MDWTWRILFLVAAATGAHS, and the GLP-1 analog is a DNA sequence encoding GLP-1(7-37 / A8G), as detailed in Table 1. [Figure 1C]

[0043] Figure 2 shows another example of a single promoter shuttle vector used to prepare recombinant adenovirus or adenoviral vectors expressing GLP-1 according to the present invention. [Figure 2A] An example of construction of a dual promoter shuttle plasmid is shown. [Figure 2B] An example of the DNA sequence of the dual promoter shuttle vector AdV-GLP-1(7-36)-CMV / EF1a is shown, which contains the CMV and EF1a promoters and encodes GLP-1(7-36). [Figure 2C] An example of the DNA sequence of the dual promoter shuttle vector AdV-GLP-1(7-36)-PLGF2-CMV / EF1a is shown, which contains the CMV and EF1a promoters. [Figure 2D] An example of the DNA sequence of the dual promoter shuttle vector AdV-PLGF2-GLP1(7-36)-CMV / EF1a is shown, which contains the CMV and EF1a promoters. [Figure 2E] Another example of constructing a dual promoter shuttle plasmid is shown below. [Figure 3A]1 shows the evaluation of the expression and secretion functions of the recombinant adenovirus expressing GLP1(7-36) described in Example II by ELISA. [Figure 3B] In the Examples, it is shown that the viral replication ability of a recombinant adenovirus (Ad5_G01) expressing GLP1 in pancreatic cancer cells is higher than that of a control adenovirus (Ad5_con) that does not express GLP1. [Figure 3C] In the Examples, it is shown that the viral replication ability of a recombinant adenovirus expressing GLP1 (Ad5_G01) in colon cancer cells is higher than that of a control adenovirus not expressing GLP1 (Ad5_con). [Figure 4] Example II shows the biologically active form of GLP1 produced by a recombinant adenovirus expressing a GLP1 analog (ADV GLP1(7-37 / A8G)). [Figure 5] 1 shows the biologically active form of GLP1 produced by recombinant adenovirus expressing GLP1(7-36) in Example II. [Figure 6] Example III(A) shows the experimental design for evaluating recombinant adenovirus expressing GLP1(7-36) in a mouse model of pancreatic cancer. [Figure 7A] 6 shows tumor growth data for the PBS negative control group in the pancreatic cancer mouse model shown in FIG. 6 (see Example III(A)). [Figure 7B] 1 shows tumor growth data for a group treated with a control adenovirus (AdV-Ctrl) that does not express the target peptide in a pancreatic cancer mouse model (shown in FIG. 6 and see Example III(A)). [Figure 7C] 1 shows tumor growth data for a group treated with recombinant adenovirus AdV-GLP1(7-36) expressing GLP1(7-36) in a pancreatic cancer mouse model (shown in FIG. 6 and see Example III(A)). [Figure 8]In a pancreatic cancer mouse model (shown in Figure 6 and see Example III(A)), the survival rate of the group treated with a recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)) was significantly increased (p<0.05) compared to the group treated with a control adenovirus (AdV-Ctrl) that does not express the target peptide and the negative control group treated with phosphate-buffered saline (PBS) alone. [Figure 9] In a pancreatic cancer mouse model (shown in Figure 6 and see Example III(A)), there was no significant difference in body weight between the group treated with a recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)), the group treated with a control adenovirus that does not express the target peptide (AdV-Ctrl), and the negative control group treated with phosphate-buffered saline (PBS) alone. [Figure 10] 1 shows the experimental design for the evaluation of recombinant adenovirus expressing GLP1(7-36) (see Example III(A)) in an orthotopic pancreatic cancer mouse model. [Figure 11A] In an orthotopic Panc02-car mouse model (shown in FIG. 10 and see Example III(A)), total luciferase counts are shown among a group treated with a recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)), a group treated with a control adenovirus that does not express the target peptide (AdV-Ctrl), and a negative control group treated with phosphate-buffered saline (PBS) only. [Figure 11B] This figure shows a comparison of tumor volume measured by luminescence in an orthotopic Panc02-car mouse model (shown in Figure 10 and see Example III(A)) between a group treated with a recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)), a group treated with a control adenovirus that does not express the target peptide (AdV-Ctrl), and a negative control group treated with phosphate-buffered saline (PBS) alone. [Figure 12]In an orthotopic Panc02-car mouse model (shown in Figure 10 and see Example III(A)), the survival rate of the group treated with a recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)) was significantly increased (p<0.05) compared to the group treated with a control adenovirus that does not express the target peptide (AdV-Ctrl) and the negative control group treated with phosphate-buffered saline (PBS) alone. [Figure 13] 1 shows the experimental design for evaluation of recombinant adenovirus expressing GLP1(7-36) (see Example III(B)) in a mouse model of colon cancer. [Figure 14A] 1 shows tumor growth data for a negative control group treated with phosphate buffered saline (PBS) alone in a colon cancer mouse model (shown in FIG. 13 and see Example III(B)). [Figure 14B] 1 shows tumor growth data for a group treated with a control adenovirus (AdV-Ctrl) that does not express the target peptide in a colon cancer mouse model (shown in FIG. 13 and see Example III(B)). [Figure 14C] 1 shows tumor growth data for a group treated with recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)) in a colon cancer mouse model (shown in FIG. 13 and see Example III(B)). [Figure 15] In a mouse model of colon cancer (shown in Figure 13 and see Example III(B)), the survival rate was significantly increased in the group treated with a recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)) compared to the group treated with a control adenovirus that does not express the target peptide (AdV-Ctrl) (p<0.01) and the negative control group treated with phosphate-buffered saline (PBS) alone (p<0.05). [Figure 16]In a mouse model of colon cancer (shown in Figure 13 and see Example III(B)), there was no significant difference in body weight between the group treated with a recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)), the group treated with a control adenovirus that does not express the target peptide (AdV-Ctrl), and the negative control group treated with phosphate-buffered saline (PBS) alone. [Figure 17] 1 shows the experimental design for evaluation of recombinant adenovirus expressing GLP1(7-36) (see Example III(C)) in a melanoma mouse model. [Figure 18] In a melanoma mouse model (shown in FIG. 17 and see Example III(C)), the survival rate was significantly increased in the group treated with recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)) compared to the group treated with a control adenovirus not expressing the target peptide (AdV-Ctrl) (P=0.0014) and the negative control group treated with phosphate-buffered saline (PBS) alone (P=0.0015). [Figure 19] 1 shows the experimental design for evaluation of recombinant adenovirus expressing GLP1(7-36) (see Example III(D)) in a mouse glioma model. [Figure 20] In a mouse glioma model (shown in Figure 19), the tumor growth rate was significantly reduced in the group treated with recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)) compared to the group treated with a control adenovirus not expressing the target peptide (AdV-Ctrl) (p=0.0001) and the negative control group treated with phosphate-buffered saline (PBS) alone (p=0.0001). [Figure 21]In a mouse glioma model (shown in Figure 19 and see Example III(D)), the survival rate was significantly increased in the group treated with recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)) compared to the group treated with a control adenovirus not expressing the target peptide (AdV-Ctrl) (p=0.0027) and the negative control group treated with phosphate-buffered saline (PBS) alone (p=0.0082). [Figure 22] 1 shows the experimental design for the evaluation of recombinant adenovirus (e.g., I(E)) expressing GLP1(7-36) in a mouse model of kidney cancer. [Figure 23A] 2 shows tumor growth data for a negative control group treated with phosphate buffered saline (PBS) alone in a kidney cancer mouse model (shown in FIG. 22 and see Example III(E)). [Figure 23B] 2 shows tumor growth data for a group treated with a control adenovirus, AdV-Ctrl, which does not express the target peptide, in a mouse kidney cancer model (shown in FIG. 22 and see Example III(C)). [Figure 23C] 2 shows tumor growth data for a group treated with recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)) in a renal cancer mouse model (shown in FIG. 22 and see Example III(C)). [Figure 23D] In a mouse kidney cancer model (shown in Figure 22), the tumor growth rate was significantly reduced in the group treated with recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)) compared to the group treated with a control adenovirus that does not express the target peptide (AdV-Ctrl) (p<0.0001) and the negative control group treated with phosphate-buffered saline (PBS) alone (p<0.05). [Figure 24]In a pancreatic cancer mouse model (see Example IV(A)), the group treated with AdV-GLP-1(7-36) after treatment with anti-CD8 antibody (GLP1(7-36) + anti-CD8) exhibited a slower tumor growth rate compared to the negative control group treated with phosphate-buffered saline (PBS) alone, the group treated with control adenovirus (AdV-Ctrl), and the groups treated with anti-CD4 antibody (GLP1(7-36) + anti-CD4) or anti-NK (GLP1(7-36) + anti-NK) before treatment with AdV-GLP-1(7-36)-CMV / EFla, respectively. [Figure 25] In a pancreatic cancer mouse model (see Example IV(A)), the survival rate of the group treated with anti-CD8 antibody (GLP1(7-36) + anti-CD8) prior to AdV-GLP-1(7-36)-CMV / EFla treatment was significantly higher than that of the group treated with control adenovirus (AdV-Ctrl), but not statistically significant, compared with the group treated with AdV-GLP-1(7-36)-CMV / EFla alone (AdV-GLP1(7-36)). The survival rate was significantly reduced, and the group treated with AdV-GLP-1(7-36)-CMV / EFla alone (AdV-GLP1(7-36)) and the group treated with anti-CD4 antibody (GLP1(7-36) + anti-CD4) or anti-NK antibody (GLP1(7-36) + anti-NK) before treatment with AdV-GLP-1(7-36)-CMV / EFla had significantly higher survival rates than the group treated with control adenovirus (AdV-Ctrl) alone (*: p<0.05) and that the survival rate of the group treated with anti-CD4 antibody (GLP1(7-36) + anti-CD4) or anti-NK antibody (GLP1(7-36) + anti-NK) before treatment with AdV-GLP-1(7-36)-CMV / EFla was not significantly different from that of the group treated with AdV-GLP-1(7-36)-CMV / EFla alone (AdV-GLP1(7-36)). [Figure 26A] This shows the percentage of interferon-γ-positive CD8-positive T cells (IFN-γ+CD8+T cells) in the AdV-GLP1(7-36) group, AdV-Ctrl group, and PBS group in a pancreatic cancer mouse model (see Example IV(B)). [Figure 26B]This shows the percentage of granzyme B-positive CD8-positive T cells (GZMB+CD8+T cells) in the AdV-GLP1(7-36) group, AdV-Ctrl group, and PBS group in a pancreatic cancer mouse model (see Example IV(B)). [Figure 26C] This shows the percentage of PD1-positive CD8-positive T cells (PD1+CD8+T cells) in the AdV-GLP1(7-36) group, AdV-Ctrl group, and PBS group in a pancreatic cancer mouse model (see Example IV(B)). [Figure 27A] 1 shows the LCN2 expression levels in CD8+ T cells in the AdV-GLP1(7-36) group, AdV-Ctrl group, and PBS group in a pancreatic cancer mouse model (see Example IV(B)). [Figure 27B] 1 shows the LCN2 expression levels in CD4+ T cells in the AdV-GLP1(7-36) group, AdV-Ctrl group, and PBS group in a pancreatic cancer mouse model (see Example IV(B)). [Figure 28A] FIG. 1 shows LCN2 expression levels in the presence or absence of GLP-1(7-36) when cultured CD8-positive T cells were co-cultured with Panc02 cells in a pancreatic cancer mouse model (see Example IV(C)). [Figure 28B] FIG. 1 shows LCN2 expression levels in the presence or absence of GLP-1(7-36) and liraglutide when cultured CD8+ T cells were co-cultured with Panc02 cells in a pancreatic cancer mouse model (see Example IV(C)). [Figure 29] AdV-GLP-1(7-36)-CMV / EFla (CD8-Panc02-AdV-GLP1(7-36)) significantly suppressed neutrophil recruitment mediated by CD8+ T cells, and significant differences were observed in in vitro experiments compared with the control adenovirus (AdV-Ctrl)-treated group (CD8-Panc02-AdV-Ctrl) (see Example IV(C)). [Figure 30] We demonstrate that recombinant adenovirus expressing GLP1(7-37 / A8G) exerts oncolytic effects on non-small cell lung cancer cells. [Figure 31] 1 shows the experimental design for evaluating recombinant adenovirus expressing GLP1(7-37 / A8G) in a mouse model of pancreatic cancer. [Figure 32] In a pancreatic cancer mouse model (e.g., as shown in Figure 31), the tumor growth rate was significantly reduced in the group treated with recombinant adenovirus expressing GLP1(7-36 / A8G) (AdV-GLP-1(7-36 / A8G)) compared to the group treated with a control adenovirus (AdV-Ctrl) that did not express the target peptide (P<0.0001) and the negative control group treated with phosphate-buffered saline (PBS) alone. [Figure 33] This shows that in a pancreatic cancer mouse model (shown in Figure 31 and see Example V(B)), the survival rate of the group treated with recombinant adenovirus expressing GLP1(7-36 / A8G) (AdV-GLP1(7-36 / A8G)) was significantly increased (p<0.05) compared to the group treated with control adenovirus (AdV-Ctrl) that does not express the target peptide. [Figure 34] In a pancreatic cancer mouse model (shown in Figure 31 and see Example V(B)), there is no significant difference in body weight between the recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36 / A8G)), the control adenovirus not expressing the target peptide (AdV-Ctrl), and the negative control group treated with phosphate-buffered saline (PBS) alone. [Figure 35] 1 shows the experimental design for evaluation of recombinant adenoviruses expressing GLP1(7-36)-PLGF2 or PLGF2-GLP1(7-36) in a mouse model of pancreatic cancer (see Example VI). [Figure 36A] 3 shows tumor growth data for a negative control group treated with phosphate buffered saline (PBS) only in a pancreatic cancer mouse model (shown in FIG. 35 and see Example VI). [Figure 36B] 3 shows tumor growth data for groups treated with recombinant adenovirus expressing GLP1(7-36)-PLGF2 (AdV-GLP1) in a pancreatic cancer mouse model (shown in FIG. 35 and see Example VI). [Figure 36C]3 shows tumor growth data for groups treated with recombinant adenovirus expressing PLGF2-GLP1(7-36) (AdV-GLP1) in a pancreatic cancer mouse model (shown in FIG. 35 and see Example VI). DETAILED DESCRIPTION OF THE INVENTION

[0010] Glucagon-like peptide-1 (GLP-1), a 30-amino acid polypeptide, is a glucagon-like peptide-1 receptor agonist that not only lowers blood glucose but also significantly suppresses multiple tumor-promoting inflammatory pathways, including the NF-κB / IL-6 / STAT3 pathway and the formation of the NLRP3 / IL-1β inflammasome.

[0011] The present invention provides a recombinant adenovirus (e.g., adenovirus subtype C Ad5) or adenoviral vector comprising a first promoter, an E1A early activation replication element, an optional second promoter, and a nucleotide (e.g., DNA) sequence encoding a target protein comprising a glucagon-like peptide-1 (GLP-1) receptor agonist. The first promoter is a constitutive promoter, and the optional second promoter is also a constitutive promoter, which may be the same as or different from the first promoter. The present invention also provides production and applications of the recombinant adenovirus or adenoviral vector.

[0012] As shown in some examples, the present invention has produced various Ad5 adenoviruses or adenoviral vectors by encoding target proteins, including GLP-1 receptor agonists, via nucleotide (e.g., DNA) sequences upstream of one or two promoters. These viruses are capable of expressing target proteins both in vivo and in vitro, and have demonstrated tumor growth inhibitory effects in vivo and in vivo against multiple tumors, including non-small cell lung cancer, pancreatic cancer, colon cancer, melanoma, glioma, and kidney cancer. In some cases, tumor regression effects have also been observed in vivo, without any significant side effects.

[0013] Furthermore, at least one exemplary Ad5 adenovirus or adenoviral vector contains a nucleotide (e.g., DNA) sequence encoding GLP-1(7-37), which induces a CD8+ T cell-mediated antitumor effect in pancreatic cancer, but not a natural killer (NK) cell-mediated antitumor effect in hepatocellular carcinoma induced by intratumoral injection of liraglutide (a GLP-1 analog). (Reference: Xian Lu, Chun Xu, Jie Dong, Shuguang Zuo, Hailin Zhang, Chunping Jiang, Junhua Wu, Jiwu Wei, Liraglutide activates natural killer cell-mediated antitumor responses by inhibiting IL-6 / STAT3 signaling in hepatocellular carcinoma, Translational Oncology, Volume 14, Issue 1, 2021, 100872)

[0014] I. Recombinant adenovirus or adenoviral vector and pharmaceutical composition thereof One aspect of the present invention relates to a recombinant adenovirus or adenoviral vector comprising a first promoter, an E1A early activation replication element, an optional second promoter, and a nucleotide (e.g., DNA) sequence encoding a target protein comprising a glucagon-like peptide-1 (GLP-1) receptor agonist, wherein the first promoter is a constitutive promoter, and the optional second promoter is also a constitutive promoter and may be the same as or different from the first promoter. In some embodiments, the nucleotide (e.g., DNA) sequence further comprises a DNA sequence encoding an interleukin-2 (IL-2) signal peptide to promote extracellular secretion of the target peptide. In some embodiments, when the target protein is GLP-1(7-36), the recombinant adenovirus or adenoviral vector comprises two promoters. In some embodiments, the E1A early activation replication element may be upstream or downstream of the nucleotide (e.g., DNA) sequence, and both the E1A early activation replication element and the nucleotide (e.g., DNA) sequence may be between the first promoter and the poly(A) tail. For example, see Figures 1A, 1C, 2A and 2E, where the first promoter is CMV.

[0015] Examples of the recombinant adenovirus include, but are not limited to, adenovirus subtype C Ad5.

[0016] Some recombinant adenoviruses (e.g., Ad5) or adenoviral vectors disclosed herein can be used to treat or prevent one or more tumors in a subject. In some embodiments, the recombinant adenoviruses (e.g., Ad5) or adenoviral vectors can induce CD8+ T cell-mediated anti-tumor effects in a subject. In some embodiments, the recombinant adenoviruses (e.g., Ad5) or adenoviral vectors can induce tumor regression in a subject.

[0017] Some recombinant adenoviruses (e.g., Ad5) or adenovirus vectors disclosed herein contain one or more constitutive promoters, selected from the CMV promoter and the EF1a promoter. The constitutive promoters may be the same or different. For example, a recombinant adenovirus (e.g., Ad5) or adenovirus vector may use a CMV promoter as both the first and second promoters; a recombinant adenovirus (e.g., Ad5) or adenovirus vector may simultaneously use an EF1a promoter as the first and second promoters; a recombinant adenovirus (e.g., Ad5) or adenovirus vector may use a CMV promoter as the first promoter and an EF1a promoter as the second promoter; or, conversely, a recombinant adenovirus (e.g., Ad5) or adenovirus vector may use an EF1a promoter as the first promoter and a CMV promoter as the second promoter.

[0018] Some recombinant adenoviruses (e.g., Ad5) or adenovirus vectors disclosed in the present invention contain two constitutive promoters and a DNA sequence encoding GLP-1(7-36), where the amino acid sequence of GLP-1(7-36) is SEQ ID NO:1.

[0019] Examples of glucagon-like peptide-1 receptor agonists include, but are not limited to, an amino acid sequence having 80% or more homology with GLP-1, an active fragment of GLP-1 (e.g., GLP-1(7-36) (SEQ ID NO:1), GLP-1(7-37)), or a GLP-1 analog (e.g., GLP-1(7-37 / A8G) (SEQ ID NO:2) having one or more mutations). Some recombinant adenoviruses (e.g., Ad5) or adenoviral vectors disclosed in the present invention contain a secreted glucagon-like peptide-1 protein as a GLP-1 receptor agonist. Examples of GLP-1 analogs include, but are not limited to, GLP-1 fragments having one or more substituted, deleted, and / or additional amino acids, and all of these GLP-1 analogs are GLP-1 receptor agonists.

[0020] In some embodiments, the target protein comprises a fusion protein of a tumor-homing protein (e.g., PLGF2) and a GLP-1 receptor agonist. In some embodiments, the fusion protein includes a linking peptide (e.g., a 4GS linker, e.g., (GGGGS)n, where n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) that links the tumor-homing protein (e.g., PLGF2) to the GLP-1 receptor agonist. In some embodiments, the GLP-1 receptor agonist is proximal to the N-terminus and the tumor-homing protein (e.g., PLGF2) is proximal to the C-terminus of the fusion protein. In some embodiments, the tumor-homing protein (e.g., PLGF2) is proximal to the N-terminus and the GLP-1 receptor agonist is proximal to the C-terminus of the fusion protein.

[0021] Examples of fusion proteins include, but are not limited to, GLP-1(7-36)-4GS-PLGF2 (e.g., SEQ ID NO:3, where n=3, applied to a 4GS linker, Table 1), PLGF2-4GS-GLP-1(7-36) (e.g., SEQ ID NO:4, where n=3, applied to a 4GS linker, Table 1), GLP-1(7-37)-4GS-PLGF2, PLGF2-4GS-GLP-1(7-37), GLP-1(7-37 / A8G)-4GS-PLGF2, and PLGF2-4GS-GLP-1(7-37 / A8G).

[0022] The recombinant adenovirus (e.g., Ad5) or adenoviral vector disclosed in the present invention comprises one or two constitutive promoters and a DNA sequence encoding a target protein, details of which are summarized in Table 1 (Figures 1A-2D).

[0023] [Table 1]

[0024] Another aspect of the present invention relates to a pharmaceutical composition comprising a recombinant adenovirus (e.g., Ad5) or adenoviral vector disclosed herein and a pharmaceutically acceptable carrier, adjuvant, excipient, or combination thereof.

[0025] Examples of pharmaceutically acceptable carriers include physiologically acceptable solutions such as sterile saline and sterile buffered saline.

[0026] Pharmaceutically acceptable carriers may contain minor amounts of excipients, such as substances that enhance isotonicity and chemical stability. These excipients should be non-toxic at dosages and concentrations applicable to humans or other mammalian subjects. Examples of such excipients include buffers (e.g., phosphate, citric acid, succinic acid, acetic acid, and other organic acids and their salts), antioxidants (e.g., ascorbic acid), low molecular weight (e.g., less than about 10 residues) polypeptides (e.g., polyarginine and tripeptides), proteins (e.g., serum albumin, gelatin, and immunoglobulins), amino acids (e.g., glycine, glutamic acid, aspartic acid, and arginine), monosaccharides, disaccharides, and other carbohydrates (e.g., cellulose and its derivatives, glucose, mannose, and dextrins), chelating agents (e.g., EDTA), sugar alcohols (e.g., mannitol and sorbitol), counterions (e.g., sodium), non-ionic surfactants (e.g., polysorbates and poloxamers), antibiotics, and polyethylene glycol (PEG).

[0027] The recombinant adenovirus or adenoviral vector, or pharmaceutical composition comprising the recombinant adenovirus or adenoviral vector, disclosed herein can be stored in single-dose or multi-dose containers, such as sealed ampoules or vials, as aqueous solutions or lyophilized products.

[0028] II. Applications of recombinant adenoviruses (e.g., Ad5) or adenovirus vectors and pharmaceutical compositions thereof Another aspect of the present invention relates to the use of the recombinant adenovirus (e.g., Ad5) or adenoviral vector disclosed herein and pharmaceutical compositions thereof in anti-cancer treatment and / or prevention in a subject. In some embodiments, the anti-tumor treatment and / or prevention comprises inducing an anti-tumor effect mediated by CD8+ T cells in the subject.

[0029] In some embodiments, a method for inducing a CD8+ T cell-mediated anti-tumor effect in a subject comprises administering to the subject a therapeutically effective amount of a recombinant adenovirus (e.g., Ad5) or adenoviral vector, or a pharmaceutical composition comprising the recombinant adenovirus or adenoviral vector.

[0030] In some examples, a method of treating one or more cancers in a subject comprises administering to the subject a therapeutically effective amount of a recombinant adenovirus (e.g., Ad5) or adenoviral vector, or a pharmaceutical composition comprising the recombinant adenovirus or adenoviral vector.

[0031] Examples of the one or more cancers include, but are not limited to, liver cancer, pancreatic cancer, colon cancer, glioma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, prostate cancer, renal cancer, head and neck squamous cell carcinoma, melanoma, multiple myeloma, and lymphoma. In some embodiments, the lung cancer is non-small cell lung cancer.

[0032] In some embodiments, the cancer is a metastatic cancer.

[0033] Some anti-cancer methods disclosed in the present invention further include inhibiting tumor invasion and / or metastasis.

[0034] Some anti-cancer methods disclosed in the present invention further include one or more effects selected from the group consisting of reducing the expression level of LCN2 in CD8+ T cells and infiltrating neutrophils into tumors.

[0035] Some anti-cancer methods disclosed herein further include slowing the progression of cachexia in a subject.

[0036] Some anti-cancer methods disclosed herein further include extending the lifespan of a subject.

[0037] As used herein, an "effective amount" refers to an amount of a compound that achieves a desired effect. For example, a set of cells can be contacted with an effective amount of a compound to study its effects in vitro (e.g., in cell culture) or to achieve a desired therapeutic effect through isolated or in vivo experiments. An effective amount of a compound can also be applied to achieving a therapeutic effect in a subject, such as preventing or treating a target disease (e.g., one or more tumors), alleviating disease-related symptoms, or achieving a desired physiological effect. In this context, the effective amount of a compound is referred to as a "therapeutically effective amount," "therapeutically effective concentration," or "therapeutically effective dose." A precise effective amount or therapeutically effective dose is the amount of compound that achieves an optimal therapeutic effect for a given subject or set of cells. This amount will vary depending on several factors, including, but not limited to, the characteristics of the compound (including activity, pharmacokinetics, potency, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, overall health status, response to a particular dose, and type of drug used), the nature of the pharmaceutically acceptable carrier or carriers used in the formulation, and the route of administration. Furthermore, the effective or therapeutically effective amount will vary depending on whether the compound is administered alone or in combination with other compounds, drugs, therapies, or other treatments. Those skilled in the art of clinical and pharmacological sciences can determine the effective or therapeutic amount by routine experimentation, i.e., by monitoring the cell's or subject's response to the compound and adjusting the dose accordingly. For additional guidance, see Remington: Pharmaceutical Science and Practice, 21st ed., University of the Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins Publishing, Philadelphia, PA, 2005, which is incorporated herein by reference in its entirety.

[0038] "Treatment" or "treatment" of a disease can refer to preventing the disease, delaying the onset or rate of progression of the disease, reducing the risk of developing the disease, preventing or delaying the progression of symptoms associated with the disease, alleviating or eliminating symptoms associated with the disease, achieving complete or partial regression of the disease, or any combination of the above effects. Treatment can also refer to prevention or prophylactic treatment of the disease.

[0039] In some embodiments, the recombinant adenovirus (e.g., Ad5) or adenovirus vector and pharmaceutical composition thereof disclosed herein can be used in combination with other existing drug products, such as immune response-promoting peptides and antibacterial agents (synthetic antibacterial agents). The recombinant adenovirus (e.g., Ad5) or adenovirus vector and pharmaceutical composition thereof disclosed herein may further contain other drugs and excipients. Examples of drugs or excipients that can be used in combination with the recombinant adenovirus (e.g., Ad5) or adenovirus vector and pharmaceutical composition thereof disclosed herein include drugs that promote the entry of the recombinant adenovirus (e.g., Ad5) or adenovirus vector and pharmaceutical composition thereof into cells, and liposomes and other transfection-promoting drugs and / or excipients (e.g., fluorocarbon emulsifiers, cochleates, nanotubes, golden particles, biodegradable microspheres, and cationic polymers).

[0040] In some embodiments, the amount of active ingredient contained in a recombinant adenovirus (e.g., Ad5) or adenoviral vector and pharmaceutical composition thereof can be selected from a wide range of concentrations, such as infectious units (ifu), weight / volume percentage (w / v%), or other quantitative indicators of the amount of active ingredient, as long as it is a therapeutically or pharmaceutically effective amount. The dose of the recombinant adenovirus (e.g., Ad5) or adenoviral vector and pharmaceutical composition thereof disclosed in the present invention can be appropriately selected depending on factors such as the expected therapeutic effect, the administration method (administration route), the treatment period, the patient's age, sex, and other conditions.

[0041] In some embodiments, when a recombinant adenovirus (e.g., Ad5) or adenoviral vector is administered as an active ingredient to a human subject, the dose of the recombinant adenovirus (e.g., Ad5) or adenoviral vector is approximately about 5 x 10 per subject. 5 ~Approx. 5×10 10 Infectious units (ifu), approximately 5 × 10 6 ~Approx. 5×10 9 ifu, approximately 1 × 10 7 ~Approx. 1×109 ifu, approximately 5 × 10 7 ~Approx. 5×10 8 ifu, approximately 1 × 10 8 ~Approx. 1×10 10 ifu, approximately 5 × 10 8 ~Approx. 5×10 10 ifu, approximately 5 × 10 7 ~Approx. 5×10 11 ifu, approximately 1 × 10 8 ~Approx. 1×10 10 ifu, or approximately 1 x 10 8 ~Approx. 1×10 11 It may be an amount corresponding to ifu.

[0042] In a further embodiment, when a recombinant adenovirus (e.g., Ad5) or adenoviral vector is administered to a subject as an active ingredient, the dose can be selected from a wide range based on the amount of expressible DNA introduced into the recombinant adenovirus (e.g., Ad5) or adenoviral vector, and also depends on the strength of the transcription and translation promoters used in the transfer vector.

[0043] In some embodiments, the recombinant adenovirus (e.g., Ad5) or adenovirus vector and pharmaceutical composition thereof disclosed herein can be administered by directly injecting a recombinant adenovirus (e.g., Ad5) or adenovirus vector suspension into a target site (e.g., one or more cancer sites). The suspension can be prepared by suspending the recombinant adenovirus (e.g., Ad5) or adenovirus vector in phosphate-buffered saline (PBS) or saline. The recombinant adenovirus (e.g., Ad5) or adenovirus vector and pharmaceutical composition thereof disclosed herein can be administered one or more times. Specifically, after the initial administration, one or more additional recombinant adenovirus (e.g., Ad5) or adenovirus vectors can be administered.

[0044] In some anti-cancer methods disclosed in the present invention, the recombinant adenovirus (eg, Ad5) or adenoviral vector and pharmaceutical composition thereof can be administered as a monotherapy, an adjunct therapy, or a combination therapy.

[0045] Some anti-cancer methods disclosed herein further include one or more therapies selected from the group consisting of one or more chemotherapy and one or more radiation therapy.

[0046] In some embodiments, a therapeutically effective amount of the recombinant adenovirus (eg, Ad5) or adenoviral vector and pharmaceutical composition thereof can be administered once daily.

[0047] In some embodiments, a therapeutically effective amount of the recombinant adenovirus (eg, Ad5) or adenoviral vector and pharmaceutical composition thereof can be administered once every other day.

[0048] In some embodiments, the therapeutically effective amount of the recombinant adenovirus (e.g., Ad5) or adenoviral vector and pharmaceutical composition thereof can be administered for at least about one week, at least about two weeks, at least about three weeks, at least about four weeks, at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about eleven months, at least about twelve months, at least about one year, at least about two years, at least about three years, at least about four years, or at least about five years. In some embodiments, the therapeutically effective amount of the recombinant adenovirus (e.g., Ad5) or adenoviral vector and pharmaceutical composition thereof can be administered until one or more cancers in the subject have substantially regressed or disappeared.

[0049] III. Production of Recombinant Adenovirus (e.g., Ad5) or Adenoviral Vectors and Pharmaceutical Compositions Thereof Another aspect of the present invention relates to the production of recombinant adenovirus (e.g., Ad5) or adenovirus vectors. One example of producing a recombinant adenovirus (e.g., Ad5) or adenovirus vector as described in the present invention includes the steps of i) constructing a shuttle plasmid, ii) virus rescue, and iii) virus propagation.

[0050] In one embodiment, constructing a shuttle plasmid includes producing a shuttle plasmid containing nucleotides (e.g., DNA) encoding a target protein, wherein the target protein includes a GLP-1 receptor agonist disclosed herein, such as, but not limited to, GLP-1, a GLP-1 fragment (e.g., GLP-1(7-36), GLP-1(7-37)), and a GLP-1 analog (e.g., a GLP-1 fragment with one or more substituted amino acids, one or more missing amino acids, and / or one or more additional amino acids). In some embodiments, the target protein includes a fusion protein of a GLP-1 receptor agonist and another protein (e.g., PLGF2).

[0051] The following examples are intended to illustrate various embodiments of the present invention. Therefore, the specific embodiments described should not be considered as limiting the scope of the present invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications are possible without departing from the scope of the present invention. It should also be understood that these equivalent embodiments are intended to be included in the present invention. Furthermore, all references cited in this specification are incorporated herein by reference as if set forth in their entirety.

[0052] IV. Examples The following example illustrates the summary of the Ad5 example as follows:

[0053] [Table 2]

[0054] EXAMPLE I. Production of Recombinant Ad5 Adenovirus or Adenovirus Vectors with Single or Dual Promoters Examples of recombinant Ad5 adenoviruses or adenoviral vectors with single or dual promoters are produced in three steps: i) construction of the Ad5 shuttle plasmid, ii) virus rescue, and iii) virus propagation, as shown below.

[0055] I(A). Construction of Ad5 shuttle plasmid A DNA sequence encoding the desired element (e.g., an example of a recombinant adenovirus with a single CMV promoter: Homologous A-CMV-E1A-P2A-GLP1(7-36)-Homologous B, see Figures 1A-1B; an example of a recombinant adenovirus with a CMV promoter as the first promoter and an EF1 promoter as the second promoter: Homologous A-CMV-E1A-EF1-GLP1(7-36)-Homologous B, see Figures 2A-2D) was synthesized in vitro. A shuttle plasmid (e.g., pDC316 AdMax shuttle) was cleaved with two restriction enzymes (e.g., XbaI and HindIII), and the synthesized DNA sequence was inserted into the plasmid in the presence of In-Fusion enzyme. The DNA sequence encoding the target protein, i.e., the shuttle plasmid containing the target DNA, was confirmed by DNA sequencing (see Table 1 for an example).

[0056] I(B). Ad5 virus rescue 293T cells were seeded into T25 flasks and incubated with pBHG-lox-(Δ)E1E3-Cre and the Ad5 shuttle plasmid containing the target DNA at 37°C in 5% CO2. After 7-10 days, the cells were collected in a 15 mL centrifuge tube (recovered by spraying with 1 mL of medium), freeze-thawed twice, and centrifuged at 3000 rpm for 15 minutes. The viral supernatant was collected and stored at -80°C as a viral stock.

[0057] I(C) Ad5 virus propagation 50 μL of virus stock solution was added to a 10 cm culture dish containing 60% 293T cells and cultured at 37°C in 5% CO2. Cells were harvested when cytopathic effects appeared. Virus was collected as described above and purified by cesium chloride density gradient centrifugation. Virus titer was measured using the TCID50 method.

[0058] Example II. Viral Functional Evaluation of Recombinant Ad5 Adenovirus Examples II(A). Expression and secretory function of GLP1 A549 cells were cultured in a 6-well plate (8 × 10 5 Cells were inoculated into 1000 cells / well and cultured overnight in DMEM containing 10% FBS. The medium was then replaced with 2% FBS-containing DMEM (1 mL / well). Recombinant Ad5 adenovirus (Example I, MOI = 100, 10, 1, 0.1, 0.01, or 0) containing target DNA (GLP-1(7-36)) was added. Supernatants were collected 48 and 72 hours after viral infection. GLP-1 expression and secretory function were measured by ELISA (Figure 3A; the bars on the left of each MOI indicate the GLP-1(7-36) concentration in samples 48 hours after viral infection, and the bars on the right indicate the GLP-1(7-36) concentration in samples 72 hours after viral infection).

[0059] Pancreatic cancer cells (Panc02) and colon cancer cells (MC38) were infected at the same MOI with a recombinant Ad5 virus containing the target DNA (Example I) and a control virus (Ad5 con) containing only the viral replication element E1A sequence, rather than the target DNA sequence. Cells were harvested at different time points. Virus suspensions were obtained by repeated freeze-thawing and centrifugation. Virus titers were measured in 293T cells, and changes in viral replication ability were analyzed (Figures 3B-3C). The results showed that the viral replication ability of the recombinant adenovirus expressing GLP1 (Ad5_G01, Figures 3B-3C) in pancreatic cancer and colon cancer cells was higher than that of the control adenovirus not expressing GLP1 (Ad5_con, Figures 3B-3C).

[0060] II(B). After A11A cell line was infected with recombinant Ad5 adenovirus, the target protein and liraglutide exhibited significant biological activity. A11A is a cell line stably expressing a fusion reporter gene for monitoring the biological activity of GLP-1. HEK293 cells (4.7 × 10 5 ~5.2×10 5 Cells were seeded into 6-well plates (1 / well) and cultured overnight at 37°C, 5% CO2. Recombinant Ad5 adenoviruses containing target DNA encoding the target protein (e.g., AdV-GLP-1(7-37 / A8G) and AdV-GLP-1(7-36)-CMV / EFla, prepared according to Example 1) were added. Cells were infected at the desired MOI (e.g., MOI = 15). 48-72 hours after viral infection, the cells and the liquid in the wells were collected separately and stored at -70°C as GLP-1 receptor agonist samples for A11A cell treatment. A negative control sample was prepared in the same manner, except that the control virus Ad5_con was added instead of the recombinant Ad5 adenoviruses AdV-GLP-1(7-37 / A8G) and AdV-GLP-1(7-36)-CMV / EFla. A11A cells were seeded into 96-well plates (5 x 10 cells / well) and cultured overnight at 37 °C, 5% CO. 100 μL of GLP-1 receptor agonist samples, negative control samples, or liraglutide (1,000 nM, 100 μL) was added to the A11A cells, and the fluorescence intensity was measured after 4 hours of incubation. The biological activity of the GLP-1 agonist in the GLP-1 receptor agonist samples is shown (Table 3 and Figures 4-5).

[0061] [Table 3]

[0062] Example III. In vivo antitumor effect of recombinant Ad5 adenovirus AdV-GLP-1(7-36)-CMV / EFla AdV-GLP-1(7-36)-CMV / EF1 was prepared according to the method of Example I(B), where the target protein sequence is SEQ ID NO: 1 and the target DNA sequence is SEQ ID NO: 5. Viral replication, GLP-1 receptor agonist expression, and its biological activity were verified according to the method of Example II (see Figure 5).

[0063] III(A). In vivo antitumor effect of AdV-GLP-1(7-36)-CMV / EFla in pancreatic cancer animal models III(A)(i) Panc02-car subcutaneous animal model Select 6-8 week old C57BL / 6 mice and inoculate 1 x 10 Panc02-car cells (suspended in 10% Matrigel in PBS) into each mouse. 6 A total of 18 mice (see Figure 6) were inoculated with 100 cells to construct subcutaneous tumor models. 3 When the number of infected mice reached 10, a negative control group was injected with PBS (0.1 mL) alone, and an Ad5 control virus (AdV-Ctrl, 5 × 10 8 a control Ad5 group injected with 5 × 10 ibu / dose of Ad5 virus containing target DNA (AdV-GLP-1(7-36)-CMV / EF1a, 5 × 10 8 The mice were randomly divided into three treatment groups, each receiving an intratumoral injection of 100 mg / kg of 1000 mg ...

[0064] Compared with the control Ad5 group (p<0.0001, Figure 7B) and the negative control group injected with PBS only (p<0.0001, Figure 7A), the AdV-GLP-1(7-36)-CMV / EFla-treated group showed a slower tumor growth rate (Figure 7C) and a statistically significant prolongation of survival (p<0.05 compared with the control Ad5 group and the negative control group, Figure 8). There was no significant change in the body weight of mice in the three groups (Figure 9).

[0065] V(A)(ii) Orthotopic pancreatic cancer model Tumors were grown in the pancreas of mice. Because in situ pancreatic tumors cannot be measured with calipers like subcutaneous tumors, tumor growth was monitored by luminescence. Significant differences are indicated in the figures with * (p<0.05).

[0066] 6-8 week old C57BL / 6 mice were selected and transfected with Panc02-car cells (suspended in 10% Matrigel in PBS, 1 × 10 cells per mouse). 6 A total of 18 mice (see Figure 10) were inoculated with 100 cells each to establish an in situ pancreatic cancer model. 3 When the number of infected mice reached 10, a negative control group was injected with PBS (0.1 mL) alone, and an Ad5 control virus (AdV-Ctrl, 1.5 × 10 9 a control Ad5 group injected with 1.5 × 10 ibu / dose of Ad5 virus containing target DNA (AdV-GLP-1(7-36)-CMV / EFla, 1.5 × 10 9 The mice were randomly divided into three groups: one receiving an intratumoral injection of IFU / IFU. All treatments were administered by a single intratumoral injection (see Figure 10). Tumor volume was measured by luminescence, and the survival time of the mice after natural death was recorded.

[0067] Compared with the control Ad5 group (p=0.0032, AdV-Ctrl, see Figures 11A-11B) and the negative control group injected with PBS only (p=0.0002, PBS, see Figures 11A-11B), the AdV-GLP-1(7-36)-CMV / EF1a-treated group showed a slower tumor growth rate (see Figures 11A-11B) and a statistically significant prolongation of survival time (p<0.05 compared with the control Ad5 group and the negative control group, see Figure 12).

[0068] III(B) In vivo antitumor effect of AdV-GLP-1(7-36)-CMV / EFla in a colon cancer animal model Six- to eight-week-old C57BL / 6 mice were selected and inoculated with MC38-car cells (10% Matrigel suspended in PBS, 1 × 10 cells per mouse) into the right flank. 6 A total of 21 mice (see Figure 13) were inoculated with 100 cells to construct a subcutaneous tumor model. 3When the number of infected mice reached 10, a negative control group was injected with PBS (0.1 mL) alone, and an Ad5 control virus (AdV-Ctrl, 5 × 10 8 a control Ad5 group injected with 5 × 10 ibu / dose of Ad5 virus containing target DNA (AdV-GLP-1(7-36)-CMV / EF1, 5 × 10 8 The mice were randomly divided into three treatment groups, each receiving an intratumoral injection of 100 mg / kg of 1000 mg ...

[0069] Compared with the control Ad5 group (p<0.0001, Figure 14B) and the negative control group injected with PBS only (p<0.0001, Figure 14A), the AdV-GLP-1(7-36)-CMV / EFla-treated group showed a statistically significantly slower tumor growth rate (Figure 14C) and a statistically significantly longer survival time (compared to the control Ad5 group, p<0.01; compared to the negative control group, p<0.05, Figure 15). There was no significant change in the body weight of mice in the three groups (Figure 16).

[0070] III(C) In vivo antitumor effect of AdV-GLP-1(7-36)-CMV / EFla in melanoma animal models Select 6-8 week old C57BL / 6 mice and inoculate each mouse with 1 x 10 B16 cells (suspended in 10% Matrigel in PBS). 6 A total of 21 mice (see Figure 17) were inoculated with 100 cells to construct a subcutaneous tumor model. 3 When the number of infected mice reached 10, a negative control group was injected with PBS (0.1 mL) alone, and an Ad5 control virus (AdV-Ctrl, 5 × 10 8 a control Ad5 group injected with 5 × 10 ibuprofen / dose) and an Ad5 virus containing target DNA, AdV-GLP-1(7-36)-CMV / EF1a, 5 × 10 8 The mice were randomly divided into three treatment groups, each receiving an intratumoral injection of 100 mg / kg of 1000 mg ...

[0071] III(D) In vivo antitumor effect of AdV-GLP-1(7-36)-CMV / EFla in animal models of glioma Select 6-8 week old C57BL / 6 mice and inoculate GL261 glioma cells (10% Matrigel suspended in PBS, 1 × 10 cells per mouse). 6 A total of 11 mice (see Figure 19) were inoculated with 100 cells to construct a subcutaneous tumor model. 3 When the number of infected mice reached 100, a negative control group was injected with PBS (0.1 mL, n = 2) alone, and an Ad5 control virus (AdV-Ctrl, 5 × 10 8 a control Ad5 group injected with 100 mg / dose (n = 4) and an Ad5 virus containing target DNA (AdV-GLP-1(7-36)-CMV / EF1a, 5 × 10 8 The mice were randomly divided into three treatment groups, each receiving an intratumoral injection of 1000 mg / kg of IgG (pfu / dose, n=5). All treatments were administered intratumorally every other day for a total of three doses (see Figure 19). The tumor volume and survival time of the mice were recorded after natural death.

[0072] Compared with the control Ad5 group (p = 0.0001, AdV-Ctrl, see Figure 20) and the negative control group injected with PBS only (p = 0.0001, PBS, see Figure 20), the AdV-GLP-1(7-36)-CMV / EFla-treated group showed a statistically slower tumor growth rate (see Figure 20) and a statistically significantly longer survival time (compared to the control Ad5 group, p = 0.0027; compared to the negative control group, p = 0.0082, see Figure 21).

[0073] III(E). In vivo antitumor effect of AdV-GLP-1(7-36)-CMV / EFla in renal cancer animal models Balb / c mice aged 6-8 weeks were selected and Renca cells (10% Matrigel suspended in PBS, 1 × 10 cells per mouse) were injected into the right flank. 6 A total of 24 mice (see Figure 22) were inoculated with 100 cells on day 0 to establish a subcutaneous tumor model. The tumor volume reached approximately 100 mm on day 10. 3When the number of infected mice reached 10, a negative control group was injected with PBS (0.1 mL) alone, and an Ad5 control virus (AdV-Ctrl, 5 × 10 8 The mice were randomly divided into three groups: a control Ad5 group injected with 1000 pfu / dose (1000 mg / dose) and a treatment group injected with Ad5 virus containing target DNA (AdV-GLP-1(7-36)-CMV / EF).

[0074] The tumor growth rate in the recombinant adenovirus AdV-GLP-1(7-36)-CMV / EF1a-treated group was significantly lower than that in the control group (AdV-Ctrl, Figures 23B and 23D) and the negative control group treated with PBS alone (Figures 23A and 23D). Specifically, the tumor growth rate in the AdV-GLP-1(7-36)-CMV / EF1a-treated group was significantly reduced compared to the control group (*p<0.001), and also showed a significant difference compared to the negative control group (*p<0.05).

[0075] Example IV: CD8 of recombinant adenovirus AdV-GLP-1(7-36)-CMV / EF1a + T cell-mediated tumor effects The recombinant adenovirus AdV-GLP-1(7-36)-CMV / EF1a was produced according to the method of Example I, and its target protein sequence is SEQ ID NO: 1 and its target DNA sequence is SEQ ID NO: 5. The replication ability of the virus, expression of the GLP-1 receptor agonist, and biological activity of the expressed GLP-1 receptor agonist were verified according to the method of Example II.

[0076] IV(A): AdV-GLP-1(7-36)-CMV / EF1a therapy in combination with anti-CD8, anti-CD4, or anti-NK therapy Pancreatic cancer cells were inoculated subcutaneously into the right axilla of C57BL / 6 mice. 3 When the tumor size reached 100 μg / mL, the mice were randomly divided into several treatment groups: intratumoral injection of PBS (PBS group, 0.1 mL / injection), control adenovirus (AdV-Ctrl group, 5 × 10 8Intratumoral injection of AdV-GLP-1(7-36)-CMV / EF1a (5 × 10 infectious units / injection), intraperitoneal injection of anti-CD8 antibody, anti-CD4 antibody, or anti-NK antibody, followed by AdV-GLP-1(7-36)-CMV / EF1a (5 × 10 8 The mice were intratumorally injected with 1000 infectious units (10 ...

[0077] The tumor growth rates of the group receiving AdV-GLP-1(7-36)-CMV / EF1a treatment alone (AdV-GLP1(7-36); Figure 24), and the groups receiving prior treatment with an anti-CD4 antibody (GLP1(7-36) + anti-CD4; Figure 24) or an anti-NK antibody (GLP1(7-36) + anti-NK; Figure 24) before AdV-GLP-1(7-36)-CMV / EF1a treatment were all significantly slower than those of the group receiving prior treatment with an anti-CD8 antibody (GLP1(7-36) + anti-CD8; Figure 24) before AdV-GLP-1(7-36)-CMV / EF1a treatment, the control adenovirus group (AdV-Ctrl; Figure 24), and the negative control group receiving only PBS treatment (PBS; Figure 24).

[0078] The group that received anti-CD8 antibody (GLP1(7-36)+anti-CD8, Figure 25) prior to AdV-GLP-1(7-36)-CMV / EF1a treatment showed no significant difference in survival rate compared to the control adenovirus group (AdV-Ctrl, Figure 25), but its survival rate was significantly reduced compared to the group that received only AdV-GLP-1(7-36)-CMV / EF1a treatment (AdV-GLP1(7-36), Figure 25).

[0079] The group receiving AdV-GLP-1(7-36)-CMV / EF1a treatment alone (AdV-GLP1(7-36); Figure 25) and the group receiving prior anti-CD4 antibody (GLP1(7-36)+anti-CD4; Figure 25) or anti-NK antibody (GLP1(7-36)+anti-NK; Figure 25) treatment before receiving AdV-GLP-1(7-36)-CMV / EF1a treatment had significantly higher survival rates than the control adenovirus group (AdV-Ctrl; Figure 25; *: *p<0.5). However, the group previously treated with anti-CD4 antibody (GLP1(7-36) + anti-CD4, Figure 25) or anti-NK antibody (GLP1(7-36) + anti-NK, Figure 25) showed no significant difference in survival rate compared with the group treated with AdV-GLP-1(7-36)-CMV / EF1a alone (AdV-GLP1(7-36), Figure 25).

[0080] IV(B): AdV-GLP-1(7-36)-CMV / EF1a treatment significantly enhanced antitumor activity and CD8 + Reduces T cell exhaustion Pancreatic cancer cells were inoculated subcutaneously into the right axilla of C57BL / 6 mice. Tumor volumes were approximately 200 mm 3 When the tumor size reached 100 mg / kg, mice were randomly divided into treatment groups, each of which received an intratumoral injection of: AdV-GLP-1(7-36)-CMV / EF1a (5 × 10 8 infectious units / dose), control adenovirus (AdV-Ctrl, 5 × 10 8 Each group received two doses, one every other day. 48 hours after the second dose, tumor tissue was isolated and analyzed for specific markers (e.g., IFN-γ). + CD8 + T cells, Figure 26A, GZMB + CD8 + T cells, Fig. 26B, PD1 + CD8 +T cells (Figure 26C). Each experimental group included five mice. The results showed a significant difference between the AdV-GLP-1(7-36)-CMV / EF1a treatment group (AdV-GLP1(7-36), Figures 26A-26C) and the control adenovirus group (AdV-Ctrl, Figures 26A-26C).

[0081] Pancreatic cancer cells were inoculated subcutaneously into the right axilla of C57BL / 6 mice. 3 When the tumor size reached 100 μg / injection, the mice were randomly divided into several treatment groups and received intratumoral injections of PBS (PBS group, 0.1 mL / injection), intratumoral injections of control adenovirus (AdV-Ctrl group, 5 × 10 catarrhal infectious units / injection), intraperitoneal injections of anti-CD8 antibody (200 μg), anti-CD4 antibody (200 μg), or anti-NK antibody (200 μg), followed by intratumoral injections of AdV-GLP-1(7-36)-CMV / EF1a (5 × 10 8 Intratumoral injections were administered twice, once every other day. Tumor volume and survival were then monitored. Each experimental group included 6-8 mice.

[0082] T cells were isolated from mouse tumor tissue (taken from samples 48 h after AdV-GLP-1(7-36)-CMV / EF1a, control adenovirus, or PBS treatment) and phenotyped by flow cytometry (FAScan).

[0083] Lipid carrier protein 2 (Lipocalin 2, LCN2), also known as Neutrophil Gelatinase-Associated Lipocalin (NGAL), is a secreted protein of the lipid carrier protein superfamily. LCN2 is involved in the development of various inflammatory diseases. Studies have shown that LCN2 promotes neutrophil recruitment and CD8 + AdV-GLP-1(7-36)-CMV / EF1a treatment suppresses the activity of CD8 T cells. +Significantly reducing LCN2 expression levels in T cells (*p < 0.05, AdV-GLP1(7-36), Figure 27A) led to increased expression of CD8 + Control adenovirus treatment (AdV-Ctrl, Figure 27A) significantly enhanced the anti-tumor response of CD8 T cells. + LCN2 expression levels in T cells were significantly increased (***p<0.0001 compared with the PBS group). In contrast, CD4 + In T cells, there was no significant difference in LCN2 expression levels between the AdV-GLP-1(7-36)-CMV / EF1a treatment group (AdV-GLP1(7-36), Figure 27B) and the control adenovirus group (AdV-Ctrl, Figure 27B).

[0084] IV(C): GLP-1(7-36) or GLP-1 analogs (lyral peptides) were co-cultured with Panc02 cells. + Significantly reduces LCN2 expression in T cells Panc02 cells were cultured with CD8 + The experimental group was cultured with T cells for 24 hours, and GLP-1(7-36) was added to the experimental group. Then, CD8 + The expression level of LCN2 in T cells was measured (Fig. 28A). The results showed that the presence of GLP-1(7-36) was associated with the expression of CD8 + It significantly reduced LCN2 expression levels in T cells (**p<0.01, PANCO-2-GLP1, Figure 28A), and inhibited Panc02 cells and CD8 + There was a significant difference compared to the control group in which only T cells were co-cultured (PANCO-2, Figure 28A). Panc02 cells were cultured in DMEM medium for 48 hours, and then the supernatant was collected and CD8 + The experimental groups were treated with either GLP-1 (7-36) or a GLP-1 analog (lyral peptide), and then CD8 T cells were detected by flow cytometry. +The expression level of LCN2 in T cells was measured (Fig. 28B). The results showed that the presence of GLP-1(7-36) (***p<0.001, CD8-GLP1, Fig. 28B) or the lyral peptide (***p<0.001, CD8-GLP1, Fig. 28B) significantly increased the expression of LCN2 in CD8 T cells. + It significantly reduced LCN2 expression levels in T cells and inhibited Panc02 and CD8 + There was a significant difference compared to the control group (CD8, Figure 28B) in which only T cells were co-cultured. Of note, the effect of GLP-1(7-36) and the lyral peptide on CD8 + There was no significant difference in their role in reducing LCN2 expression levels in T cells (Figure 28B).

[0085] IV(D):AdV-GLP-1(7-36)-CMV / EF1a is CD8 + Significantly suppressed T cell-mediated neutrophil recruitment Panc02 cells were infected with AdV-GLP-1(7-36)-CMV / EF1a, control adenovirus (AdV-Ctrl), or PBS and cultured for 24 hours. CD8 cells isolated from tumor tissue were then cultured. + The cells were cultured with CD8 T cells for 24 hours. + A T cell suspension was added to the lower chamber of the Transwell cell, and neutrophils isolated from mice were added to the upper chamber. After 4 hours, the cell suspension in the lower chamber was collected and the Ly6G neutrophil marker was detected (Figure 29).

[0086] Compared with the control adenovirus treatment group (***p<0.001, CD8-Panc02-AdV-Ctrl, Figure 29), the AdV-GLP-1(7-36)-CMV / EF1a treatment group (CD8-Panc02-AdV-GLP1(7-36), Figure 29) showed significantly higher CD8 + It significantly inhibited T cell-mediated neutrophil recruitment.

[0087] Previous studies have shown that intratumoral injection of GLP-1 analogs (lyral peptides) can induce antitumor effects via natural killer (NK) cells, and have shown remarkable effects in hepatocellular carcinoma models. However, in this study, the recombinant adenovirus AdV-GLP-1(7-36)-CMV / EF1a (prepared according to Example I) expressing GLP-1(7-36) did not induce antitumor effects via NK cells, but rather via CD8 in a pancreatic cancer model. + AdV-GLP-1(7-36)-CMV / EF1a induced antitumor effects via CD8 T cells. + The adenovirus-treated group significantly reduced LCN2 expression in T cells and significantly reduced intratumoral neutrophil infiltration, demonstrating that intratumoral neutrophil infiltration may exert immunosuppressive effects in certain solid tumors.

[0088] Example V: In vitro antitumor effect of recombinant Ad5 adenovirus ADV-GLP-1(7-37 / A8G)-CMV According to the method of Example I(A), AdV-GLP-1(7-37 / A8G)-CMV was produced, whose target protein sequence is SEQ ID NO:2 and whose target DNA sequence is SEQ ID NO:6. The viral replication ability, expression of the GLP-1 receptor agonist, and its biological activity were verified according to the method of Example II.

[0089] V(A): In vitro antitumor effect of AdV-GLP-1(7-37 / A8G)-CMV on non-small cell lung cancer cells A549 lung cancer cells were infected with AdV-GLP-1(7-37 / A8G)-CMV and control adenovirus (Ad5) at multiplicities of infection (MOI) of 1 to 100. 72 hours after infection, cell viability was measured by the MTT assay to evaluate the oncolytic effect of Ad5-GLP1.

[0090] VII(B): In vivo antitumor effect of AdV-GLP-1(7-37 / A8G)-CMV in pancreatic cancer animal models Six- to eight-week-old C57BL / 6 mice were selected to establish a right axillary subcutaneous tumor model (Panc02 cells were suspended in PBS containing 10% Matrigel, and 1 × 10 cells were injected into each mouse). 6 A total of 18 mice were inoculated with 100 cells (Figure 31). 3 When the mice reached the target size, they were randomly divided into three groups: a negative control group (PBS only, 0.1 mL injection), a control adenovirus group (AdV-Ctrl, 5 × 10 7 infectious units / dose), treatment group (AdV-GLP-1(7-37 / A8G)-CMV, 5 × 10 7 Infectious units per injection). All treatment groups received three intratumoral injections, one every other day (Figure 31). Tumor volume and mouse weight were then monitored, and survival time after natural death was recorded.

[0091] Compared with the control adenovirus group (AdV-Ctrl, p<0.001, Figure 32), the tumor growth rate in the AdV-GLP-1(7-37 / A8G)-CMV treatment group was significantly reduced (AdV-GLP1(7-37 / A8G), Figure 32). Compared with the negative control group injected with PBS only (PBS, Figure 32), the tumor growth rate in the AdV-GLP-1(7-37 / A8G)-CMV treatment group was also significantly reduced. Furthermore, the survival rate of the AdV-GLP-1(7-37 / A8G)-CMV-treated group (AdV-GLP1(7-37 / A8G), Figure 33) was significantly improved compared to the control adenovirus group (AdV-Ctrl, p<0.05, Figure 33). The survival rate of the AdV-GLP-1(7-37 / A8G)-CMV-treated group was also improved compared to the negative control group injected with PBS alone (PBS, Figure 33). There was no significant change in the body weight of mice in the three groups (Figure 34).

[0092] Example VI: In vivo antitumor effects of recombinant Ad5 adenovirus AdV-GLP-1(7-36)-4GS-PLGF2-CMV / EF1a and AdV-PLGF2-4GS-GLP-1(7-36)-CMV / EF1a AdV-GLP-1(7-36)-4GS-PLGF2-CMV / EF1a and AdV-PLGF2-4GS-GLP-1(7-36)-CMV / EF1a were prepared according to the method of Example I(B). The target protein sequence of AdV-GLP-1(7-36)-4GS-PLGF2-CMV / EF1a is SEQ ID NO:3, the target DNA sequence is SEQ ID NO:7, and the target protein sequence of AdV-PLGF2-4GS-GLP-1(7-36)-CMV / EF1a is SEQ ID NO:4, the target DNA sequence is SEQ ID NO:8. The viral replication ability, GLP-1 receptor agonist expression, and biological activity were verified according to the method of Example II.

[0093] Six- to eight-week-old C57BL / 6 mice were selected to establish a right axillary subcutaneous tumor model (panc02-car cells were suspended in PBS containing 10% Matrigel, and 1 × 10 cells were injected into each mouse). 6 A total of 15 mice were inoculated with 100 cells (Figure 35). The tumor volume was approximately 100 mm 3 When the tumor size reached 100 μg / mL, the mice were randomly divided into three groups: a negative control group (injected with PBS only, Figure 36A), a control adenovirus group (AdV-Ctrl), and a treatment group (AdV-GLP-1(7-36)-4GS-PLGF2-CMV / EF1a, Figure 36B or AdV-PLGF2-4GS-GLP-1(7-36)-CMV / EF1a, Figure 36C). Mice in each group received three intratumoral injections (5 × 10 8 Infectious units / dose or 0.1 mL PBS) and tumor volume was monitored (Figure 35).

[0094] Compared with the negative control group injected with PBS only (Figure 36A), the tumor growth rates in both the AdV-GLP-1(7-36)-4GS-PLGF2-CMV / EF1a (Figure 36B) and AdV-PLGF2-4GS-GLP-1(7-36)-CMV / EF1a (Figure 36C) treatment groups were significantly reduced.

Claims

1. a first promoter, an ElA early activation replication element, an optional second promoter, and a target nucleotide sequence encoding a target protein comprising a glucagon-like peptide-1 (GLP-1) receptor agonist; the first promoter is a constitutive promoter, The recombinant adenovirus or adenoviral vector, wherein the optional second promoter is a constitutive promoter that is the same as or different from the first promoter.

2. 2. The recombinant adenovirus or adenoviral vector of claim 1, wherein the constitutive promoter is selected from the group consisting of a CMV promoter and an EF1a promoter.

3. 2. The recombinant adenovirus or adenoviral vector of claim 1, wherein the first promoter is a CMV promoter.

4. 2. The recombinant adenovirus or adenoviral vector of claim 1, wherein the second promoter is an EF1a promoter.

5. 2. The recombinant adenovirus or adenoviral vector of claim 1, further comprising a nucleotide sequence encoding an IL-2 signal peptide.

6. The recombinant adenovirus or adenovirus vector of claim 1, wherein the GLP-1 receptor agonist comprises an amino acid sequence having 80% or more homology with a sequence selected from the group consisting of biologically active fragments of GLP-1 (e.g., GLP-1(7-36), GLP-1(7-37)) and GLP-1 analogs.

7. The recombinant adenovirus or adenoviral vector of claim 1 , wherein the target proteins include a GLP-1 receptor agonist and a tumor-homing protein.

8. The GLP-1 receptor agonist and the tumor-homing protein are GGGGS ｎ 2. The recombinant adenovirus or adenoviral vector of claim 1 , wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

9. The recombinant adenovirus or adenoviral vector of claim 6, wherein the GLP-1 analogue is a GLP-1 fragment in which one or more amino acids have been substituted, deleted and / or added.

10. The recombinant adenovirus or adenoviral vector of claim 1, wherein the GLP-1 receptor agonist is a secreted glucagon-like peptide-1 protein.

11. A recombinant adenovirus or adenovirus vector as described in claim 1, which produces one or more effects selected from tumor growth inhibition, tumor regression, and survival extension in one or more tumors with which the recombinant adenovirus or adenovirus vector comes into contact.

12. When administered to a subject, it increases the CD8 ＋ The recombinant adenovirus or adenovirus vector of claim 1, which induces an anti-tumor effect mediated by T cells.

13. A pharmaceutical composition comprising the recombinant adenovirus or adenovirus vector described in claim 1 and a pharmaceutically acceptable carrier.

14. CD8 of the subject ＋ The pharmaceutical composition of claim 13, which is for use in a method for inducing a T cell-mediated anti-tumor effect.

15. The pharmaceutical composition of claim 13, for use in a method for treating or preventing one or more cancers in a subject.

16. 16. The pharmaceutical composition of claim 15, wherein the one or more cancers are selected from the group consisting of liver cancer, pancreatic cancer, colon cancer, glioma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, prostate cancer, renal cancer, head and neck squamous cell carcinoma, melanoma, multiple myeloma and lymphoma.

17. The pharmaceutical composition of claim 16, wherein the lung cancer is non-small cell lung cancer.

18. A pharmaceutical composition described in claim 14 or 15, wherein the method of inducing an anti-tumor effect or the method of treating or preventing cancer further comprises inhibiting tumor invasion and / or metastasis.

19. The method for inducing an antitumor effect or the method for treating or preventing cancer comprises administering to a subject a CD8 ＋ 16. The pharmaceutical composition of claim 14 or 15, further comprising one or more effects selected from the group consisting of reducing LCN2 expression in T cells, reducing intratumoral infiltration of neutrophils, inhibiting tumor inflammatory pathways, inhibiting IDO-1 and / or restoring anti-cancer immune monitoring.

20. A pharmaceutical composition described in claim 14 or 15, wherein the method of inducing an anti-tumor effect or the method of treating or preventing cancer further comprises delaying the progression of cachexia in the subject.

21. A pharmaceutical composition described in claim 14 or 15, wherein the method of inducing an anti-tumor effect or the method of treating or preventing cancer further comprises extending the lifespan of the subject.

22. 16. The pharmaceutical composition of claim 14 or 15, wherein the pharmaceutical composition is administered as a monotherapy, adjunctive therapy or combination therapy.

23. The pharmaceutical composition according to claim 14 or 15, wherein the pharmaceutical composition is administered intratumorally.

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